Method for detecting content of fluoride ions in microsamples

ABSTRACT

A method for detecting content of fluoride ions in a microsample, including: (1) preparing reagents; (2) preparing a detecting liquid; (3) loading samples; and (4) detecting the content of fluoride ions. The method of the invention can effectively overcome unstable factors in the prior art. Moreover, this method involves simple and rapid operation, the use of a small amount of a sample and simultaneous detection of multiple samples. The steps are standardized to reduce human error, allowing for more reliable results.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority from Chinese Patent Application No. 201811039399.9, filed on Sep. 6, 2018. The content of the aforementioned application, including any intervening amendments thereto, is incorporated herein with reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to chemical detection, and particularly to a method for detecting the content of fluoride ions in microsamples, more specifically to a method for detecting fluoride ions in water or a liquid microsample which is colorless and transparent after pretreatment.

BACKGROUND OF THE INVENTION

For the detection of content of fluoride ions in water, the National Standard discloses several methods, including: spectrophotometry, ion selective electrode method, ion chromatography and visual colorimetry with zirconium alizarinsulfonate.

Regarding the spectrophotometry, an alizarin complexone and a lanthanum nitrate are introduced to react with the fluoride to form a blue complex, of which the color intensity is linearly correlated with the content of fluoride ions in a certain range. This method is performed by a spectrophotometer. The defects of this method include large single sample amount, requirement of manual operation and strict control of pH during the preparation of the detection liquid.

In the ion selective electrode method, the lanthanum fluoride monocrystal shows selectivity to fluoride ions, resulting in a potential difference between different concentrations of fluorine solutions on both sides of the lanthanum fluoride film. This potential difference is generally called a film potential, which is related to the ion activity of the fluoride solution. The fluorine electrode and the saturated calomel electrode constitute a pair of galvanic batteries, and the concentration of fluoride ions in the water sample can be directly determined according to the linear relationship between the potential and the negative logarithm of the ion activity. This method requires a fluoride ion selective electrode, a saturated calomel electrode, a precision pH-meter and an electromagnetic stirrer. However, this method involves disadvantages of large single sample amount, complicated and time-consuming detection process and susceptibility to ions and temperature.

As for the ion chromatography, the target anion in the water sample is eluted into the ion exchange system with a system of carbonate and bicarbonate. Since various anions have their respective retention times in the analytical column, the target anion can be separated and determined. The separated anions are converted into carbonic acid with high electrical conductivity after passing through cation exchange column or a suppressor system, such that various anions in the water sample can be simultaneously analyzed. The conductivity of individual anion components is measured by a conductivity detector, where the relative retention time and the peak height or area are respectively used for qualification and quantification. An ion chromatograph instrument is necessary for this method. However, this method still has some defects such as high requirement for the instrument, complicated operation and requirement of manual operation.

With respect to the visual colorimetry with zirconium alizarinsulfonate, sodium alizarine sulfonate reacts with a zirconium salt under an acidic condition to form a red complex, while in the presence of fluoride ions, a colorless zirconium fluoride is formed to discolor the solution, which can be quantified by visual colorimetry. However, this method prefers relatively pure water, which is not suitable for practical application.

However, there are many unstable factors during the implementation of the above methods. For example, during the operation, there may be difference in reagents of different batches; and the manual operation may result in some interference in the preparation of a detection liquid, the adjustment of pH of a buffer solution and the drawing of a standard curve. Therefore, a certain difference may exist in the results obtained by different laboratories and even in the results obtained by different people in the same laboratory. Given the above, there is an urgent need to improve the above methods.

SUMMARY OF THE INVENTION

An object of the invention is to provide a complete set of a method for detecting content of fluoride ions in a microsample to overcome the unstable factors in the prior art, allowing for more standardized, unified and reliable detection.

The invention provides a method for detecting content of fluoride ions in a microsample, comprising:

(1) preparing a reagent A, a reagent B, a reagent C, a reagent D, a reagent E and a fluoride standard solution with a concentration of 2.5 mg/L;

wherein, the reagent A is an analytical acetone; the reagent B is an analytical diacetone; the reagent C is a reagent having a pH of 5.0 and containing 0.05 mol/L of alizarin complexone; the reagent D is a reagent having a pH of 4.1 and containing sodium acetate; and the reagent E is a reagent having a pH of 4.1 and containing 0.05 mol/L of lanthanum nitrate;

(2) mixing 7 parts by volume of the reagent A, 2 parts by volume of the reagent B, 2 parts by volume of the reagent C and 2 parts by volume of the reagent D uniformly followed by mixing with 2 parts by volume of the reagent E to obtain a detecting liquid for use;

(3) selecting 8 wells in a 96-well microplate as main wells, adding different amounts of the fluoride standard solution to the main wells followed by dilution to 100 μL with deionized water; wherein concentrations of the fluoride ions in the main wells are 0 mg/L in well A, 0.039 mg/L in well B, 0.078 mg/L in well C, 0.156 mg/L in well D, 0.313 mg/L in well E, 0.625 mg/L in well F, 1.25 mg/L in well G and 2.5 mg/L in well H, respectively;

selecting some wells from the rest wells of the 96-well microplate as secondary wells followed by addition of 100 μL of a sample to respective wells; and

(4) adding 150 μL of the detecting liquid prepared in step (2) to respective main wells for complete reaction; after 15 min, transferring the microplate to a microplate reader; measuring the absorbance at 650 nm within 60 min; plotting a standard curve of fluoride ion according to the absorbance and the fluoride ion concentrations in respective main wells to calculate concentrations of fluoride ions in the samples in respective secondary wells.

In an embodiment, the reagent C is prepared by a method, comprising the following steps:

adding 1.927 g of the alizarin complexone to a 100 mL beaker; adding 5 mL of deionized water by a micropipette; then dropwise adding 5-15 mL of 1 mol/L sodium hydroxide solution to dissolve the alizarin complexone; after the alizarin complexone is dissolved, adding 0.625 g of sodium acetate to produce a mixture, and adjusting the mixture to pH 5.0 with 1 mol/L hydrochloric acid followed by dilution to 100 mL with deionized water to produce the reagent C containing 0.05 mol/L of the alizarin complexone.

In an embodiment, the reagent D is prepared by a method, comprising the following steps:

dissolving 3.5 g of sodium acetate in 80 mL of deionized water; adding 7.5 mL of glacial acetic acid followed by dilution to 100 mL with deionized water to produce a mixture; then adjusting the mixture to pH 4.1 with an acetic acid or sodium hydroxide solution to obtain the reagent D.

In an embodiment, the reagent E is prepared by a method, comprising the following steps:

weighing 2.215 g of solid lanthanum nitrate; dropwise adding 3-8 mL of a hydrochloric acid solution to dissolve the solid lanthanum nitrate to produce a mixture; and adjusting the mixture to pH 4.1 with 1 mol/L sodium acetate followed by dilution to 100 mL with deionized water to obtain the reagent E containing 0.05 mol/L of the lanthanum nitrate.

In an embodiment, the loading process specifically comprises the steps of:

adding 200 μL of the 2.5 mg/L fluoride standard solution to the H well and adding 100 μL of deionized water to each of the rest main wells; transferring 100 μL of the 2.5 mg/L fluoride standard solution in the well H to the well G followed by mixing to produce a mixture G; accurately transferring 100 μL of the mixture G to the well F followed by mixing to produce a mixture F; treating the rest main wells sequentially in the same manner; and discarding 100 μL of the mixture B instead of transferring it to the well A.

Compared to the prior art, the invention has the following beneficial effects.

1. This invention develops a new detection method to avoid deviations caused by human factors during the preparation of the detecting liquid and the buffer solution, enabling a more standardized, unified and reliable detection.

2. The traditional methods for detecting content of fluoride ions in water generally involve the use of a large amount of the sample (generally greater than 10 mL for a single sample) and the reagent (generally greater than 10 mL for a single sample). Moreover, these methods cannot enable simultaneous detection of multiple samples and require a long time for detection (generally more than 5 min for a single sample). Therefore, these methods fail to enable the miniaturization and kitization of the accurate, rapid, small-sample and high-throughput detection. However, a method involving microscale, rapid and high-throughput detection for content of fluoride ions in water is developed herein by selecting appropriate instrument, detection wavelength, concentration of the fluoride standard solution, concentration and formulation of the detecting liquid, protective agent and buffer solution.

DETAILED DESCRIPTION OF EMBODIMENTS

The invention is further described below with reference to the embodiments, and the advantages and features of the invention will become more apparent. However, the embodiments are merely exemplary, which are not intended to limit the scope of the invention. It should be understood that any modifications and replacements made by those skilled in the art without departing from the spirit and scope of the invention should still fall within the scope of the invention.

Example 1

A fluoride standard solution having a known concentration of 0.5 mg/L was used herein as sample 1, and 8 main wells were used to plot a standard curve and 20 secondary wells were used for the detection of sample 1.

(1) Preparation of Reagents

A reagent A, a reagent B, a reagent C, a reagent D, a reagent E and a fluoride standard solution having a concentration of 2.5 mg/L were prepared, where the reagent A was an analytical acetone; the reagent B was an analytical diacetone; the reagent C was a reagent having a pH of 5.0 and containing 0.05 mol/L of alizarin complexone; the reagent D was a reagent having a pH of 4.1 and containing sodium acetate; and the reagent E was a reagent having a pH of 4.1 and containing 0.05 mol/L of lanthanum nitrate.

The reagent C was prepared by the following steps.

1.927 g of alizarin complexone was weighed in a 100 mL beaker, added with 5 mL of deionized water by a micropipette, and then dropwise added with 5-15 mL of 1 mol/L sodium hydroxide solution to dissolve the alizarin complexone. After the alizarin complexone was dissolved, 0.625 g of sodium acetate was added to produce a mixture. The mixture was adjusted to pH 5.0 with 1 mol/L hydrochloric acid and diluted to 100 mL with deionized water to produce the reagent C containing 0.05 mol/L of the alizarin complexone.

The reagent D was prepared by the following steps.

3.5 g of sodium acetate was dissolved in 80 mL of deionized water, added with 7.5 mL of glacial acetic acid was, diluted to 100 mL with deionized water and adjusted to pH 4.1 with an acetic acid or sodium hydroxide solution using a pH meter to produce the reagent D.

The reagent E was prepared by the following steps.

2.215 g of solid lanthanum nitrate was weighed, dropwise added with 3-8 mL of hydrochloric acid, adjusted to pH 4.1 with 1 mol/L sodium acetate and diluted to 100 mL with deionized water to produce the reagent E containing 0.05 mol/L of the lanthanum nitrate.

(2) Preparation of a Detecting Liquid 2,100 μL of the reagent A, 600 μL of the reagent B, 600 μL of the reagent C, 600 μL of the reagent D were mixed, added with 600 μL of the reagent E and completely mixed to produce the detecting liquid for use.

(3) Loading

8 wells in a 96-well microplate were selected as main wells, where the last well, i.e., the well H, was added with 200 μL of the 2.5 mg/L fluoride standard solution and the wells A-G were respectively added with 100 μL of deionized water. Then 100 μL of the fluoride standard solution in the well H was accurately transferred to the well G by a micropipette and mixed to produce a mixture Cc and 100 μL of the mixture G was accurately transferred by a micropipette to the well F and mixed to produce a mixture F. The rest main wells were sequentially treated in the same manner, until 100 μL of the mixture B was discarded instead of transferring it to the well A. Thus, the liquid in each main well was 100 μL, and fluoride ion concentrations in the wells A-H were 0 mg/L, 0.039 mg/L, 0.078 mg/L, 0.156 mg/L, 0.313 mg/L, 0.625 mg/L, 1.25 mg/L and 2.5 mg/L, respectively.

20 wells among the rest wells in the 96-well microplate were selected as secondary wells and respectively added with 100 μL of the sample 1 (a fluoride standard solution having a known concentration of 0.5 mg/L).

(4) Detection of Content of Fluoride Ions

150 μL of the detecting liquid prepared in (2) was separately added to all of the 28 wells for reaction. After 5 min, the 96-well microplate was transferred to a microplate reader, and the absorbance was measured at 650 nm within 20 min. The detection results were shown in table 1 below.

TABLE 1 Absorbance (650 nm) of the sample 1 Column 1 (main Column 2 Column 3 Column 4 ID wells) (secondary wells) (secondary wells) (secondary wells) A 0.092 0.141 0.142 0.141 B 0.096 0.142 0.142 0.143 C 0.099 0.142 0.141 0.142 D 0.107 0.142 0.142 0.141 E 0.121 0.141 0.141 n/a F 0.152 0.142 0.141 n/a G 0.212 0.143 0.142 n/a H 0.338 0.141 0.143 n/a

A standard curve was plotted according to the fluoride contents in the wells A-H in the column 1 (respectively 0.039 mg/L, 0.078 mg/L, 0.156 mg/L, 0.313 mg/L, 0.625 mg/L, 1.250 mg/L, 2.500 mg/L) and the corresponding absorbance, where the regression equation was y=0.0982x−0.0008 with a regression coefficient R² of 0.9998.

The fluoride contents in the 20 secondary wells were calculated according to the above equation and shown in Table 2.

TABLE 2 Fluoride content (mg/L) of the sample 1 Column 1 (main Column 2 Column 3 Column 4 ID wells) (secondary wells) (secondary wells) (secondary wells) A 0 0.500 0.510 0.500 B 0.039 0.510 0.510 0.520 C 0.078 0.510 0.500 0.510 D 0.156 0.510 0.510 0.500 E 0.313 0.500 0.500 n/a F 0.625 0.510 0.500 n/a G 1.250 0.520 0.510 n/a H 2.500 0.500 0.520 n/a

According to the above results, the average fluoride content of the sample 1 was calculated as 0.508 mg/L with standard deviation of 0.007 mg/L and variation coefficient of 1.38%.

Example 2

A fluoride standard solution having a concentration of 1.0 mg/L was used herein as sample 2. 8 main wells were selected for the plotting of a standard curve, and 20 secondary wells were selected for the detection of sample 2.

(1) Preparation of Reagents

Reagents were prepared in the same manner as that in Example 1.

(2) Preparation of a Detecting Liquid

2,100 μL of the reagent A, 600 μL of the reagent B, 600 μL of the reagent C, 600 μL of the reagent D were mixed, then added with 600 μL of a reagent E, and completely mixed to produce the detecting liquid for use.

(3) Loading

8 wells in a 96-well microplate were selected as main wells, where the last main well, i.e., the well H, was added with 200 μL of the 2.5 mg/L fluoride standard solution and the wells A-G were respectively added with 100 μL of deionized water. Then 100 μL of the fluoride standard solution in the well H was accurately transferred to the well G by a micropipette and mixed to produce a mixture G, and 100 μL of the mixture G was accurately transferred by a micropipette to the well F and mixed to produce a mixture F. The rest main wells were sequentially treated in the same manner, until 100 μL of the mixture B was discarded instead of transferring it to the well A. Thus, the liquid in each main well was 100 μL, and fluoride ion concentrations in the wells A-H were 0 mg/L, 0.039 mg/L, 0.078 mg/L, 0.156 mg/L, 0.313 mg/L, 0.625 mg/L, 1.25 mg/L and 2.5 mg/L, respectively.

20 wells among the rest wells in the 96-well microplate were selected as secondary wells and respectively added with 100 μL of the sample 2 (a fluoride standard solution having a known concentration of 1.0 mg/L).

(4) Detection of Content of Fluoride Ions

150 μL of the detecting liquid prepared in (2) was separately added to all of the 28 wells for reaction. After 5 min, the 96-well microplate was transferred to a microplate reader, and the absorbance was measured at 650 nm within 20 min. The detection results were shown in Table 3 below.

TABLE 3 Absorbance (650 nm) of the sample 2 Column 1 (main Column Column 3 Column 4 ID wells) (secondary wells) (secondary wells) (secondary wells) A 0.086 0.180 0.179 0.180 B 0.089 0.180 0.180 0.179 C 0.093 0.179 0.181 0.179 D 0.102 0.180 0.180 0.180 E 0.114 0.181 0.180 n/a F 0.143 0.180 0.180 n/a G 0.205 0.181 0.180 n/a H 0.321 0.181 0.181 n/a

A standard curve was plotted according to the fluoride contents in the wells A-H in the column 1 (respectively 0 mg/L, 0.039 mg/L, 0.078 mg/L, 0.156 mg/L, 0.313 mg/L, 0.625 mg/L, 1.250 mg/L and 2.500 mg/L) and the corresponding absorbance, where the regression equation was y=0.0938x−0.0009 with a regression coefficient R² of 0.9997.

According to this regression equation, the fluoride contents in the 20 secondary wells were calculated and the results were shown in Table 4.

TABLE 4 Fluoride contents (mg/L) of the sample 2 Column 1 (main Column 2 Column 3 Column 4 ID wells) (secondary wells) (secondary wells) (secondary wells) A 0.000 1.000 0.990 1.000 B 0.039 1.000 1.000 0.990 C 0.078 0.990 1.010 0.990 D 0.156 1.000 1.000 1.000 E 0.313 1.010 1.000 n/a F 0.625 1.000 1.000 n/a G 1.250 1.010 1.000 n/a H 2.500 1.010 1.010 n/a

According to the above results, the average fluoride content of the sample 2 was calculated as 1.001 mg/L with standard deviation of 0.007 mg/L and variation coefficient (CV) of 0.669%.

Example 3

A fluoride standard solution having a concentration of 1.5 mg/L was used herein as sample 3. 8 main wells were selected to plot a standard curve, and 20 secondary wells were selected for the detection of sample 3.

(1) Preparation of Reagents

Reagents were prepared in the same manner as that in Example 1. (2) Preparation of a detecting liquid

2,100 μL of the reagent A, 600 μL of the reagent B, 600 μL of the reagent C, 600 μL of the reagent D were mixed, added with 600 μL of the reagent E and completely mixed to produce the detecting liquid for use.

(3) Loading

8 wells in a 96-well microplate were selected as main wells, where the last well, i.e., the well H, was added with 200 μL of the 2.5 mg/L fluoride standard solution and the wells A-G were respectively added with 100 μL of deionized water. Then 100 μL of the fluoride standard solution in the well H was accurately transferred to the well G by a micropipette and mixed to produce a mixture and 100 μL of the mixture G was accurately transferred by a micropipette to the well F and mixed to produce a mixture F. The rest main wells were sequentially treated in the same manner, until 100 μL of the mixture B was discarded instead of transferring it to the well A. Thus, the liquid in each main well was 100 μL, and fluoride ion concentrations in the wells A-H were 0 mg/L, 0.039 mg/L, 0.078 mg/L, 0.156 mg/L, 0.313 mg/L, 0.625 mg/L, 1.25 mg/L and 2.5 mg/L, respectively.

20 wells among the rest wells in the 96-well microplate were selected as secondary wells and respectively added with 100 μL of the sample 3 (a fluoride standard solution having a known concentration of 1.5 mg/L).

(4) Detection of Content of Fluoride Ions

150 μL of the detecting liquid prepared in step (2) was separately added to all of the 28 wells for reaction. After 5 min, the 96-well microplate was transferred to a microplate reader, and the absorbance was measured at 650 nm within 20 min. The detection results were shown in Table 5.

TABLE 5 Absorbance (650 nm) of the sample 3 Column 1 (main Column 2 Column 3 Column 4 ID wells) (secondary wells) (secondary wells) (secondary wells) A 0.092 0.239 0.240 0.237 B 0.096 0.240 0.239 0.239 C 0.099 0.239 0.237 0.239 D 0.107 0.239 0.238 0.238 E 0.121 0.240 0.239 n/a F 0.152 0.238 0.240 n/a G 0.212 0.239 0.237 n/a H 0.338 0.241 0.239 n/a

A standard curve was plotted based on the fluoride contents in the A-H wells in the column 1 (respectively 0 mg/L, 0.039 mg/L, 0.078 mg/L, 0.156 mg/L, 0.313 mg/L, 0.625 mg/L, 1.250 mg/L and 2.500 mg/L) and the corresponding absorbance, where the regression equation was y=0.0982x−0.0008 with a regression coefficient R² of 0.9998.

The fluoride contents in the 20 secondary wells were calculated according to this regression equation, and the results were shown in Table 6.

TABLE 6 Fluoride contents (mg/L) of the sample 3 Column 1 (main Column 2 Column 3 Column 4 ID wells) (secondary wells) (secondary wells) (secondary wells) A 0.000 1.500 1.510 1.480 B 0.039 1.510 1.500 1.500 C 0.078 1.500 1.480 1.500 D 0.156 1.500 1.489 1.489 E 0.313 1.510 1.500 n/a F 0.625 1.489 1.510 n/a G 1.250 1.500 1.480 n/a H 2.500 1.520 1.500 n/a

According to the above results, the average fluoride content of the sample 3 was calculated as 1.498 mg/L with standard deviation of 0.011 mg/L and variable coefficient (CV) of 0.717%.

Example 4

A fluoride standard solution having a concentration of 2.0 mg/L was used herein as sample 4. 8 main wells were selected to plot a standard curve, and 20 secondary wells were selected for the detection of the sample 4.

(1) Preparation of Reagents

Reagents were prepared in the same manner as that in Example 1. (2) Preparation of a detecting liquid

2,100 μL of the reagent A, 600 μL of the reagent B, 600 μL of the reagent C and 600 μL of the reagent D were mixed, added with 600 μL of the reagent E and completely mixed to produce the detecting liquid for use.

(3) Loading

8 wells in a 96-well microplate were selected as main wells, where the last well, i.e., the well H, was added with 200 μL of the 2.5 mg/L fluoride standard solution and the wells A-G were respectively added with 100 μL of deionized water. Then 100 μL of the fluoride standard solution in the well H was accurately transferred to the well G by a micropipette and mixed to produce a mixture and 100 μL of the mixture G was accurately transferred by a micropipette to the well F and mixed to produce a mixture F. The rest main wells were sequentially treated in the same manner, until 100 μL of the mixture B was discarded instead of transferring it to the well A. Thus, the liquid in each main well was 100 μL, and fluoride ion concentrations in the wells A-H were 0 mg/L, 0.039 mg/L, 0.078 mg/L, 0.156 mg/L, 0.313 mg/L, 0.625 mg/L, 1.25 mg/L and 2.5 mg/L, respectively.

20 wells among the rest wells in the 96-well microplate were selected as secondary wells and respectively added with 100 μL of the sample 4 (a fluoride standard solution having a known concentration of 2.0 mg/L).

(4) Detection of Content of Fluoride Ions

150 μL of the detecting liquid four prepared in step (2) was separately added to all of the 28 wells for reaction. After 5 min, the 96-well microplate was transferred to a microplate reader, and the absorbance was measured at 650 nm within 20 min. The detection results were shown in Table 7.

TABLE 7 Absorbance (650 nm) of the sample 4 Column 1 (main Column 2 Column 3 Column 4 ID wells) (secondary wells) (secondary wells) (secondary wells) A 0.090 0.090 0.090 0.090 B 0.282 0.282 0.282 0.282 C 0.282 0.282 0.282 0.282 D 0.094 0.094 0.094 0.094 E 0.281 0.281 0.281 n/a F 0.283 0.283 0.283 n/a G 0.097 0.097 0.097 n/a H 0.281 0.281 0.281 n/a

A standard curve was plotted according to the fluoride contents in the wells A-H in the column 1 (respectively 0 mg/L, 0.039 mg/L, 0.078 mg/L, 0.156 mg/L, 0.313 mg/L, 0.625 mg/L, 1.250 mg/L and 2.500 mg/L) and the corresponding absorbance, where the regression equation was y=0.0965x−0.0005 with a regression coefficient R² of 1.

According to the regression equation, the fluoride contents in the 20 secondary wells were calculated and the results were shown in Table 8.

TABLE 8 Fluoride contents (mg/L) of the sample 4 Column 1 (main Column 2 Column 3 Column 4 ID wells) (secondary wells) (secondary wells) (secondary wells) A 0.000 0.000 0.000 0.000 B 2.000 2.000 2.000 2.000 C 2.000 2.000 2.000 2.000 D 0.039 0.039 0.039 0.039 E 1.989 1.989 1.989 n/a F 2.010 2.010 2.010 n/a G 0.078 0.078 0.078 n/a H 1.989 1.989 1.989 n/a

According to the above results, the average fluoride content of the sample 4 was calculated as 1.996 mg/L with standard deviation of 0.010 mg/L and variable coefficient (CV) of 0.518%.

It can be seen from the above examples that the method of the invention for detecting the fluoride ion in a microsample has desirable accuracy and reproducibility. 

What is claimed is:
 1. A method for detecting content of fluoride ions in a microsample, comprising: (1) preparing a reagent A, a reagent B, a reagent C, a reagent D, a reagent E and a fluoride standard solution with a concentration of 2.5 mg/L; wherein, the reagent A is an analytical acetone; the reagent B is an analytical diacetone; the reagent C is a reagent having a pH of 5.0 and containing 0.05 mol/L of alizarin complexone; the reagent D is a reagent having a pH of 4.1 and containing sodium acetate; and the reagent E is a reagent having a pH of 4.1 and containing 0.05 mol/L of lanthanum nitrate; (2) mixing 7 parts by volume of the reagent A, 2 parts by volume of the reagent B, 2 parts by volume of the reagent C and 2 parts by volume of the reagent D uniformly followed by mixing with 2 parts by volume of the reagent E to obtain a detecting liquid for use; (3) selecting 8 wells in a 96-well microplate as main wells, adding different amounts of the fluoride standard solution to the main wells followed by dilution to 100 μL with deionized water; wherein concentrations of the fluoride ions in the main wells are 0 mg/L in well A, 0.039 mg/L in well B, 0.078 mg/L in well C, 0.156 mg/L in well D, 0.313 mg/L in well E, 0.625 mg/L in well F, 1.25 mg/L in well G and 2.5 mg/L in well H, respectively; selecting some wells from the rest wells of the 96-well microplate as secondary wells followed by addition of 100 μL of a sample to respective wells; (4) adding 150 μL of the detecting liquid prepared in step (2) to respective main wells for complete reaction; after 15 min, transferring the microplate to a microplate reader; measuring the absorbance at 650 nm within 60 min; plotting a standard curve of fluoride ion according to the absorbance and the fluoride ion concentrations in respective main wells to calculate concentrations of fluoride ions in the samples in respective secondary wells.
 2. The method of claim 1, wherein the reagent C is prepared by a method comprising the following steps: adding 1.927 g of the alizarin complexone to a 100 mL beaker; adding 5 mL of deionized water by a micropipette; then dropwise adding 5-15 mL of 1 mol/L sodium hydroxide solution to dissolve the alizarin complexone; after the alizarin complexone is dissolved, adding 0.625 g of sodium acetate to produce a mixture, and adjusting the mixture to pH 5.0 with 1 mol/L hydrochloric acid followed by dilution to 100 mL with deionized water to produce the reagent C containing 0.05 mol/L of the alizarin complexone.
 3. The method of claim 1, wherein the reagent D is prepared by a method, comprising the following steps: dissolving 3.5 g of sodium acetate in 80 mL of deionized water; adding 7.5 mL of glacial acetic acid followed by dilution to 100 mL with deionized water to produce a mixture; then adjusting the mixture to pH 4.1 with an acetic acid or sodium hydroxide solution to obtain the reagent D.
 4. The method of claim 1, wherein the reagent E is prepared by a method, comprising the following steps: weighing 2.215 g of solid lanthanum nitrate; dropwise adding 3-8 mL of a hydrochloric acid solution to dissolve the solid lanthanum nitrate to produce a mixture; and adjusting the mixture to pH 4.1 with 1 mol/L sodium acetate followed by dilution to 100 mL with deionized water to obtain the reagent E containing 0.05 mol/L of the lanthanum nitrate. 